Elucidation of Molecular and Genetic Regulators of Plant Programmed Cell Death

Programmed cell death (PCD) is a form of genetically regulated cellular suicide that may occur as part of normal development, or in response to abiotic or biotic triggers. While initially thought to be unique to complex multicellular life, it is now known that PCD also occurs in unicellular lifeforms, and may have first evolved in prokaryotic cells. In animals, PCD has been well described, and many distinct modes of cell death have been identified by their morphological, biochemical and genetic characteristics. While some aspects of cell death induction appear conserved in plants, it is clear there are many substantial differences, and our understanding of the key molecular and genetic regulators of plant PCD remains incomplete. In plants, PCD has been shown to be an essential component of the plant response to abiotic stresses such as drought, heat-stress, high light and waterlogging. Further, plants rely on specific forms of PCD during immune responses to prevent the further spread of pathogens. Finally, PCD is an intrinsic component of multiple developmental process such as lateral root formation, embryogenesis and senescence, which contribute both to general plant fitness, and, in the case of crops, to overall yield and efficiency. Therefore, a greater understanding of plant PCD and the manner in which it is regulated is necessary in order to enhance crop security, increase productivity and safeguard food supplies in a rapidly changing climate. One of the major limitations in the study of PCD in plants is the difficulty in isolating only dying cells, which are generally surrounding by living tissue. Additionally, as cell death is a dynamic process, sampling cells at the correct point in the cell death pathway is essential to ensure that the early events underlying PCD induction can be identified. Finally, care must be taken to ensure that only cells destined to die via PCD are studied, as the inclusion of cells that survive and induce pro-survival pathways, or cells which will die in an uncontrolled ‘necrotic’ manner, may distort such analysis. To tackle this problem, this project utilised plant cell suspension cultures, which provide a homogenous population of cells that can be continuously monitored for PCD morphology using simple light microscopy. These cell cultures are also amenable to both PCD induction and inhibition using a range of chemical and physical methods. Using this approach, RNA-sequencing of cells in which PCD had been induced or blocked using multiple treatments enabled the isolation of candidate PCD regulatory genes, which were then tested and validated in planta using both established and novel cell death and stress tolerance assays in Arabidopsis. Bioinformatics analysis of PCD transcriptomes identified upstream transcriptional regulators of plant PCD, and several hallmarks of the ‘core’ transcriptional response to PCD inducing stimuli. The same experimental platform was used to established the role of polyamines in the regulation of plant PCD, revealing that they have a dose dependent effect in Arabidopsis, inducing PCD at high concentrations while lower concentrations inhibit cell death induced by both biotic and abiotic stress. Finally, plant cell suspension cultures were used to develop a system mimicking the mechanical shear stress that cells are subjected to in stirred tank bioreactors. This mechanical stress induced PCD in cell cultures, and this cell death could be inhibited via the addition of conditioned media or glutamate to the cell culture media. The results from this thesis have contributed to our understanding of the genetic and molecular basis of plant PCD regulation, and point towards ways in which this knowledge can be leveraged to aid the development of improved agricultural and bioprocessing methods.